Recent years have seen a tremendous interest in the bottom‐up reconstitution of minimal biomolecular systems, with the ultimate aim of creating an autonomous synthetic cell. One of the universal features of living systems is cell growth, where the cell membrane expands through the incorporation of newly synthesized lipid molecules. Here, the gradual tension‐mediated growth of cell‐sized (≈10 µm) giant unilamellar vesicles (GUVs) is demonstrated, to which nanometer‐sized (≈30 nm) small unilamellar vesicles (SUVs) are provided, that act as a lipid source. By putting tension on the GUV membranes through a transmembrane osmotic pressure, SUV–GUV fusion events are promoted and substantial growth of the GUV is caused, even up to doubling its volume. Thus, experimental evidence is provided that membrane tension alone is sufficient to bring about membrane fusion and growth is demonstrated for both pure phospholipid liposomes and for hybrid vesicles with a mixture of phospholipids and fatty acids. The results show that growth of liposomes can be realized in a protein‐free minimal system, which may find useful applications in achieving autonomous synthetic cells that are capable of undergoing a continuous growth–division cycle.

Thermostability is the key to maintain the structural integrity and catalytic activity of enzymes in industrial biotechnological processes, such as terpene cyclase-mediated generation of medicines, chiral synthons, and fine chemicals. However, affording a large increase in the thermostability of enzymes through site-directed protein engineering techniques can constitute a challenge. In this paper, we used ancestral sequence reconstruction to create a hyperstable variant of the ent -copalyl diphosphate synthase PtmT2, a terpene cyclase involved in the assembly of antibiotics. Molecular dynamics simulations on the μs timescale were performed to shed light on possible molecular mechanisms contributing to activity at an elevated temperature and the large 40 °C increase in melting temperature observed for an ancestral variant of PtmT2. In silico analysis revealed key differences in the flexibility of a loop capping the active site, between extant and ancestral proteins. For the modern enzyme, the loop collapses into the active site at elevated temperatures, thus preventing biocatalysis, whereas the loop remains in a productive conformation both at ambient and high temperatures in the ancestral variant. Restoring a Pro loop residue introduced in the ancestral variant to the corresponding Gly observed in the extant protein led to reduced catalytic activity at high temperatures, with only moderate effects on the melting temperature, supporting the importance of the flexibility of the capping loop in thermoadaptation. Conversely, the inverse Gly to Pro loop mutation in the modern enzyme resulted in a 3-fold increase in the catalytic rate. Despite an overall decrease in maximal activity of ancestor compared to wild type, its increased thermostability provides a robust backbone amenable for further enzyme engineering. Our work cements the importance of loops in enzyme catalysis and provides a molecular mechanism contributing to thermoadaptation in an ancestral enzyme.

The pandemic caused by Severe acute respiratory syndrome coronavirus 2 1 has had devastating consequences on global health and economy. 2 Despite the success of vaccination campaigns 3 emerging variants 4-6 are of concern and novel viruses with the potential to drive future pandemics are circulating in nature. 7,8 Development of vaccines can be challenging, as key viral protein antigens can be unstable or aggregate. In this study, we present the application of ancestral sequence reconstruction on coronavirus spike protein, resulting in stable and highly soluble ancestral scaffold antigens (AnSAs). The AnSAs interacted with plasma of patients recovered from COVID-19 but did not bind to the human angiotensin-converting enzyme 2 (ACE2) receptor. Cryo-EM analysis of the AnSAs yielded high resolution structures (2.6-2.8 Å) indicating a closed pre-fusion conformation in which all three receptor-binding domains (RBDs) are facing downwards. This captured closed state is stabilised by an intricate hydrogen-bonding network mediated by well-resolved loops, both within and across monomers, tethering the N-terminal domain and RBD together, which determines their relative spatial orientation. Finally, we show how AnSAs are potent scaffolds by replacing the ancestral RBD with the Wuhan wild-type sequence, which restored ACE2 binding and increased the interaction with convalescent plasma. In contrast to rational antigen design depending on prior structural knowledge, our work highlights how stable and potentially interesting antigens can be generated using exclusively available sequence information. MainPhylodynamic studies have shown that Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) acquires, on average, one substitution every 11 th day. 9 Several SARS-CoV-2 variants display enhanced infectiousness and/or increased mortality rates as well as evasion from immunity from vaccination or previous infection. 6 For the omicron variant BNT162b2 (Pfizer-BioNTech) vaccine effectiveness was reduced from about 90% to 70%. 10 Despite the rapid development of several vaccines against SARS-CoV-2, the lack of comprehensive protection against infection with emerging variants therefore continues to present a risk to global health care systems. Moreover, beta-coronaviruses have previously caused epidemics (severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS)), and searches in available sequence data revealed many previously unknown and potentially pathogenic coronaviruses. 8

Due to prolonged exposure, microplastic particles are found in human blood and other bodily fluids. Despite a lack of toxicity studies regarding microplastics, harmful effects for humans seem plausible and cannot be excluded. As small plastic particles readily translocate from the gut to body fluids, enzyme-based treatment of serum could constitute a promising avenue to clear synthetic polymers and their responding oligomers by degradation into monomers. Still, the activity of plastic degrading enzymes in serum remains unknown. Here we report how engineered PETases can depolymerize microplastic-like particles of the commodity polymer polyethylene terephthalate (PET) into its non-toxic monomer terephthalic acid (TPA) in human serum at 37°C. By developing an efficient method to depolymerize microplastics in vitro, our work takes a step closer to find a solution to the problem that microplastics in the bloodstream may pose in the future.